Antibodies to hepatitis A capsid proteins have been produced against purified, SDS-disrupted whole virions. In addition, Dr. Lee Maloy has prepared antibodies against a synthetic peptide representing the entire predicted 23 amino acids of VP4, and the carboxy terminus of VP2. Dr. Richard Lerner has prepared antibodies against a set of peptides spanning the entire predicted sequence of VP1. These antibodies were studied by immunoprecipitation and western blot analysis of the viral structural peptides. Because the predicted size for the capsid protein VP4 is only 23 amino acids (about 1/3rd the size of most picornavirus VP4's) we first questioned whether the cleavage of VP0 into VP2 and VP4 actually occurs. The antibody to VP4 specifically recognized a larger peptide in the 25 to 30 kDa range which is presumable VP0 (VP4 plus VP2). We could not identify a viral VP4 band in these western blots but the sensitivity of the test for synthetic VP4 was very low (approximately 1 mug of peptide). An antibody to a synthetic peptide representing the carboxy terminus of the capsid protein VP2 recognized 2 closely spaced bands (VP0 and VP2) on western blots. Thus, it appears that cleavage of VP0 to yield VP2 and VP4 occur but it is possible that the very small VP4 is lost form the mature virion. We have produced HAV "epitope libraries" in the expression vector lambda gt11. A fragment of HAV cDNA representing the entire capsid coding region was isolated. Random fragments of this DNa were produced by limited DNase digestion and these fragments were cloned into lambda gt11. These libraries were screened by neutralizing monoclonal antibodies, but specific clones were not identified. These results suggest that the epitopes recognized by these antibodies are discontinuous or conformational and a different approach will be required to identify them.